Project Summary The development of novel chemotherapeutic approaches against cariogenic biofilms is challenging. Bacteria within biofilms are enmeshed in an exopolysaccharides (EPS)-rich matrix. Furthermore, EPS-embedded bacteria also create highly protected and acidic microenvironments that promote cariogenic biofilm build-up and acid-dissolution of tooth enamel. To overcome these remarkable challenges, our previous NIH supported (DE018023) studies developed a potent anti-caries approach by combining food-derived antibiofilm agents (myricetin and farnesol) with fluoride. We demonstrated that these agents in combination severely compromise EPS-matrix assembly and cariogenic biofilm development, resulting in a highly effective anti-caries therapy in vivo. Despite promising activity, there are limitations for further development and clinical translation of this approach. Both farnesol and myricetin are insoluble in aqueous solutions. In addition, retention of these agents at tooth-biofilm interface could be enhanced to maximize their efficacy in vivo. To address these hurdles, we have developed pH-responsive nanoparticle carriers (NPC) capable of co-encapsulating myricetin (Myr) and farnesol (Far) which were completely water-soluble, important towards practical formulations for human use. Furthermore, topically applied NPC bind avidly to pellicle and EPS, and accumulate within biofilms. Excitingly, NPC respond to acidic pH to release agents more rapidly at acidic (pathological) versus neutral (physiological) pH, greatly improving (~20-fold more effective than free agents) antibiofilm activity in vitro. We hypothesize that NPC will substantially amplify the efficacy of our combination therapy (CT) via increased solubility, retention and pH-activated release of active agents with fluoride. To support our hypothesis, Aim 1 will optimize physicochemical properties of NPC to improve targeted delivery of our agents, and thereby potentiate their antibiofilm efficacy. We will focus on increasing the kinetics of NPC pH-responsive drug release to ensure maximal release of the agents at pH consistent with the acidic biofilm milieu. Then, Aim 2 will evaluate the efficacy of optimized NPCs containing Myr and Far with fluoride (CT-NPC) using our in vitro cariogenic biofilm model. We have previously identified the major biological actions (EPS synthesis and acidogenicity) and molecular targets (gtfB, atpD) of our therapy. Thus, we will investigate how CT-NPC disrupts these virulence properties more effectively than CT using novel methods to assess spatiotemporal development of EPS matrix, acidic pH niches and gene expression in situ within intact 3D biofilms. Aim 3 will evaluate the efficacy of the developed CT-NPC in disrupting cariogenic biofilms and reducing dental caries in vivo using a rodent model of dental caries under clinically-relevant topical treatment regimen. CT-NPC will be also compared to `gold standards' of caries prevention (fluoride) and antimicrobial therapy (chlorhexidine). Successful completion of these aims will lead to a highly efficacious and clinically-translatable therapy that may be superior to current anti-plaque/anti-caries modalities and will motivate formulation development for clinical studies.